The present invention relates to methods of intein-mediated ligation of proteins. More specifically, the present invention relates to intein-mediated ligation of expressed proteins containing a predetermined N-terminal residue and/or a C-terminal thioester generated via use of one or more naturally occurring or modified inteins. Preferably, the predetermined residue is cysteine.
Inteins are the protein equivalent of the self-splicing RNA introns (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)), which catalyze their own excision from a precursor protein with the concomitant fusion of the flanking protein sequences, known as exteins (reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).
Studies into the mechanism of intein splicing led to the development of a protein purification system that utilized thiol-induced cleavage of the peptide bond at the N-terminus of the Sce VMA intein (Chong et al., Gene 192(2):271-281 (1997)). Purification with this intein-mediated system generates a bacterially-expressed protein with a C-terminal thioester (Chong et al., (1997)). In one application, where it is described to isolate a cytotoxic protein, the bacterially expressed protein with the C-terminal thioester is then fused to a chemically-synthesized peptide with an N-terminal cysteine using the chemistry described for “native chemical ligation” (Evans et al., Protein Sci. 7:2256-2264 (1998); Muir et al., Proc. Natl. Acad. Sci. USA 95:6705-6710 (1998)).
This technique, referred to as “intein-mediated protein ligation” (IPL), represents an important advance in protein semi-synthetic techniques. However, because chemically-synthesized peptides of larger than about 100 residues are difficult to obtain, the general application of IPL is limited by the requirement of a chemically-synthesized peptide as a ligation partner.
IPL technology would be significantly expanded if an expressed protein with a predetermined N-terminus, such as cysteine, could be generated. This would allow the fusion of one or more expressed proteins from a host cell, such as bacterial, yeast or mammalian cells.
One method of generating an N-terminal cysteine is with the use of proteases. However, proteases have many disadvantages, such as the possibility of multiple protease sites within a protein, as well as the chance of non-specific degradation. Furthermore, following proteolysis, the proteases must be inactivated or purified away from the protein of interest before proceeding with IPL. (Xu, et al., Proc. Natl. Acad. Sci. USA 96(2):388-393 (1999) and Erlandson, et al., Chem. Biol., 3:981-991 (1996))
There is, therefore, a need for an improved intein-mediated protein ligation method which overcomes the noted limitations of current IPL methods and which eliminates the need for use of proteases to generate an N-terminal cysteine residue. Such an improved IPL method would have widespread applicability for the ligation of expressed proteins, for example, labeling of extensive portions of a protein for, among other things, NMR analysis.